Molecular biology depends for its importance on the existence of purified recombinant DNA plasmids. E.g., Thompson, BioChromatography 1:68 (1986); Current Protocols in Molecular Biology, Greene Publishing Assoc. & Wiley, 1987; and Sambrook, Fritsch, and Maniatis, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. (All patents and publications cited hereunder are incorporated herein by reference.) Of critical significance to the development of a practical technology was the discovery that cells could incorporate extrachromosomal genetic material. Consequently, independently-replicating extrachromosomal DNA, such as plasmid DNA, can be used as a vehicle for the insertion into and amplification of any given DNA segment, from a wide variety of biological sources, in a suitable cell host which will maintain this genetic material.
Advances have been made in the fermentation and tissue culture processes used for both analytical and preparative scale growth of prokaryotic and eukaryotic cells recombinant plasmid DNA. These biological processes of synthesis generate the plasmid DNA product together with a complex mixture of cellular components including lipids, carbohydrates, lipoproteins, proteins, polysaccharides, chromosomal DNA, ribosomes, RNA and other macromolecular components. It is necessary to isolate plasmid DNA in a pure form for subsequent application.
Numerous procedures have been developed independently for the purification of cellular plasmid DNA. However, all of these processes share three basic features: (1) cellular growth; (2) cellular lysis; and (3) separation of plasmid DNA from cellular RNA and DNA. Various extractions are of key importance for the accurate, reproducible purification of plasmid DNA. These extractions are employed to remove the bulk of contaminating molecules (e.g., proteins, cellular DNA, RNA, etc.), especially when applied to complex biological systems. Proper extractions are essential for both chromatography and non-chromatography processes of plasmid purification.
A step-by-step conventional protocol for purification of plasmid DNA from 1 liter (1-2 g wet weight) of E. coli cells is presented in Table 1.